Method for the detection of virus

ABSTRACT

THE PRESENCE OR ABSENCE OF VIRUS MAY BE DETECTED BY INOCULATING A TISSUE CULTURE, WHICH IS AN APPROPRIATE HOST FOR THE SUSPECTED VIRUS, WITH A SAMPLE OF THE MATERIAL SUSPECTED TO CONTAIN A VIRUS AND THEN COMPARING THE AMOUNT OF ADENOSINE TRIPHOSPHATE IN THE CULTURE BEFORE AND AFTER A PERIOD OF INCUBATION. THE ADENOSINE TRIPHOSPHATE CONTENT OF THE INOCULATED TISSUE CULTURE IS DETERMINED BY REACTING THE TISSUE CULTURE WITH A MIXTURE COMPRISING LUCIFERIN, LUCIFERASE, AND A CATION IN THE PRESENCE OF OXYGEN AND MONITORING THE AMOUNT OF BIOLUMINESCENCE GIVEN OFF THEREBY. A SIGNIFICANT CHANGE IN THE AMOUNT OF BIOLUMINESCENE AFTER INCUBATION INDICATES THE PRESENCE OF VIRUS, WHEREAS THE ABSENCE OF VIRUS IS INDICATED BY NO SIGNIFICANT CHANGE.

United States Patent 3,575,812 METHOD FOR THE DETECTION OF VIRUS EmmettW. Chappelle, Baltimore, Md., assignor to Hazleton Laboratories,Incorporated, Falls Church, Va. No Drawing. Continuation-impart ofapplication Ser. No.

739,913, June 18, 1968, which is a continuation of application Ser. No.433,462, Feb. 17, 1965. This application Nov. 7, 1968, Ser. No. 774,194

Int. Cl. C121: N04

US. Cl. 195-1035 Claims ABSTRACT OF THE DISCLOSURE The presence orabsence of virus may be detected by inoculating a tissue culture, whichis an appropriate host for the suspected virus, with a sample of thematerial suspected to contain a virus and then comparing the amount ofadenosine triphosphate in the culture before and after a period ofincubation. The adenosine triphosphate content of the inoculated tissueculture is determined by reacting the tissue culture with a mixturecomprising luciferin, luciferase, and a cation in the presence of oxygenand monitoring the amount of bioluminescence given off thereby. Asignificant change in the amount of bioluminescene after incubationindicates the presence of virus, whereas the absence of virus isindicated by no significant change.

CROSS REFERENCES TO OTHER APPLICATION It is known that ATP is present inall living cells and tissues. The energy requirements for all biologicalreactions are directly or indirectly supplied through ATP. It has beenfound that any change in cellular integrity, such as the invasion of avirus, will be reflected in the kinetics of ATP metabolism.

It is an object of this invention to provide a method for the rapiddetection of virus in a material by monitoring the ATP level of thematerial.

It is another object of this invention to utilize the bioluminescentreaction between ATP and firely lantern extract as a means for detectingthe presence or absence of a virus in a host cell.

These and other objects are attained by the practice of this inventionwhich, briefly, comprises inoculating a tissue culture, which is anappropriate host for the suspected virus, with a sample of the materialsuspected to contain a virus. The ATP content of the inoculated tissueculture is monitored both before and following an incubation period. Thepresence of a virus in the material being tested will be indicated by asignificant change (e.g. increase or decrease) in the ATP content of theinoculated tissue culture following the period of incubation since ithas been found that the ATP content of virus-containing cells isdifferent than the amount of ATP in normal cells.

In the preferred embodiment of this invention, duplicate tissue culturesare provided and one of the tissue cultures is inoculated with a sampleof the unknown material which is suspected to contain a virus. After asuitable incubation period, equal aliquots of each of the tissue3,575,812 Patented Apr. 20, 1971 cultures are mixed in the presence ofoxygen with firefly lantern extract which includes a mixture comprisingluciferin, luciferase and a cation such as magnesium. A reaction willoccur which is accompanied by the emission of light. The quantity oflight is measured and the amount of light emitted by the inoculatedtissue culture is compared with the amount of light emitted by thenoninoculated tissue culture. The presence of a virus in the unknownmaterial will be indicated by a significant change in the amount oflight emitted by the inoculated tissue culture over the non-inoculatedtissue culture on an equal aliquot basis while the absence of virus willbe indicated by no significant change. The emission of light is causedby the reaction of ATP with the constituents of the firefly lanternextract in the presence of oxygen. Since the ATP content ofvirus-containing cells is significantly different than the amount of ATPfound in normal cells, the viruscontaining cells will emit a differentamount of light when mixed with firefly lantern extract than normalcells. Thus, the practice of this embodiment of the invention utilizesthe phenomenon of firefly bioluminescence to establish the presence of avirus by the reaction of ATP with firefly lantern extract.

The practice of this invention may also be accomplished by inoculating atissue culture with a sample of the material which is suspected tocontain a virus, immediately assaying an aliquot of the tissue culturefor ATP content, and assaying an equal aliquot of the tissue culture forATP content following a suitable incubation period. A significant changein ATP content following incubation, on an equal aliquot basis, willindicate the presence of a virus in the unknown material. The ATP assaymay be conducted by the firefly bioluminescent method previouslyreferred to.

The reactants required for firefly bioluminescence are the substrate,luciferin; the enzyme, luciferase; the activator, ATP; a cation (usuallymagnesium); and oxygen. The overall reaction is an oxidation reactioncatalyzed by the enzyme, luciferase, which results in the emission oflight. A general mechanism of the course of the reaction is as follows:

Mg ATP lueiferin luciferyl adenylate pyrophosphate luciferase Thisreaction is absolutely specific for ATP. The ATP may not be replaced byany other known compound.

The firefly bioluminescent reaction may be carried out utilizing crudefirefly lantern extracts or the purified constituents therefrom whichparticipate in the bioluminescent reaction. It has been found that asufliciently high degree of sensitivity may be attained using theprimary extract of the firefly lantern.

Lyophilized firefly lantern extract may be obtained commercially. Thismaterial may be prepared for use by dissolving it in distilled,deionized water to the desired concentrations. The extract used in theexamples which follow, unless otherwise specified, were obtained bydissolving 70 mg. of lyophilized firefly lantern extract in 5 ml. ofwater. The lyophilized preparation also contains MgSO, and potassiumarsenate in amounts sufiicient to result in concentrations of 0.01 M and0.05 M, respectively. The pH of such a solution is 7.4. The solutionsmay be further diluted to give any desired concentration of fireflylantern extract.

The firefly lantern extract which may be used in the practice of thisinvention may also be prepared in the 3 laboratory from dessicatedfirefl tails. The firefly tails are first ground to a fine powder with amortar and pestle with a small amount of washed silica. The powder isthen extracted with 0.05 M potassium arsenate-0.01 M MgSo at pH 7.4.

When crude firefly lantern extract is used in the practice of thisinvention, there may be present in the extract small amounts of ATP andATP precursors, along with phosphorylating enzymes capable of convertingthe precursors into ATP. The presence of these materials may give riseto a basal light emission by the firefly extract in the absence ofexogenous ATP. This type of light emission, which is referred to aninherent light, occasionally may interfere with the detection of lightemission in the practice of this invention. However, the problem ofinherent light may be eliminated or minimized by one or more of thefollowing techniques:

(1) The firefly extract may be partially purified to remove the factorsresponsible for the inherent light. The separation and partialpurification of luciferase and luciferin is described by McElroy(Methods in Enzymology, vol. II, page 851, Academic Press, Inc., NewYork, 1955).

(2) Another approach to the removal of inherent light involves thesalting out of luciferase by the addition of ammonium sulfate to thefirefly extract leaving the nonprotein factors responsible for inherentlight in the supernatant. This has been accomplished as follows: 50 mg.of lyophilized extract were suspended in ml. of 2.7 M ammonium sulfate.After standing at room temperature for minutes, the suspension wascentrifuged at approximately 2006 for 10 minutes after which thesupernatant was discarded. The precipitate, after being washed twicewith 10 ml. aliquots of 2.7 M ammonium sulfate, was taken up in 2.5 ml.of a solution of 0.05 M potassium arsenate buffer (pl-I 7.4) and 0.01 Mmagnesium sulfate. This treatment, followed by reconstitution withpartially purified luciferin, reduces the overall activity of theextract by only about 15 percent and reduces the inherent light by about90 percent.

(3) Experiments have indicated that the use of calcium phosphate gelwill also reduce the inherent light in the extract without significantlyreducing the activity of the extract. Thus, 50' mg. of commerciallyophilized firefly extract were dissolved in 1.25 ml. of deionizedwater and centrifuged. The solution was then treated from one to threetimes with varying amounts of calcium phosphate gel (from 249 mg. to 334mg.). The treatment consisted of shaking the gel with the extract forten minutes and then removing the gel by centrifugation at 2006 for 10minutes. With one treatment with calcium phosphate gel, there is areduction of about 85 percent of the inherent light with only a loss offrom 7 to 26 percent of luciferin-luciferase activity.

(4) The simplest means for reducing the inherent light is by dilution ofthe extract with Water. Maximum sensitivity with the least amount ofinherent light is obtained at a lyophilized extract concentration of 3mg./ml.

The practice of this invention may be used to determine rapidly thepresence of a suspected virus present in small quantities of tissues.Using electronic equipment, tissues or cells containing an amount of ATPless than 2x10 ,ug. and approaching 10 ,ug, may be assayed. Thebioluminescent reaction itself takes less than 0.5 second to attainmaximum amplitude.

The tissue cultures which may be used in the practice of this inventionare commercially available. The particular tissue cultures used shouldbe appropriate hosts for the virus which is suspected to be present inthe unknown material.

Listed below are several pathogenic viruses characterized by theirsevere infectious nature and/or their frequent occurrence. Also listedare appropriate host tissue cultures fo ach virus. i

Poliomyelitis Influenza Adenovirus Variola (small pox) PsittacosisHerpes B Tissue culture HeLa cells, chicken embryo.

L cells (mouse).

Hamster kidney.

Chicken and mouse embryo. Chicken and mouse embryo.

Hamster kidney, mouse ependymoma.

Monkey kidney.

Chicken embryo.

HeLa cells, monkey kidney.

HeLa cells, chick embryo.

HeLa cells, monkey kidney.

Monkey kidney.

Monkey kidney.

Coxsackie A and B ECHO Monkey kidney, human amnion.

The tissue culture which is an appropriate host for the suspected virusis divided into equal aliquots to pr vide duplicate cultures of thetissue. One of the cultures is then inoculated with a sample of theunknown material. Both the inoculated and the non-inoculated culturesare then incubated under conditions standard for the particular tissuecultures used. After a suitable period of incubation, the time of whichwill vary with the particular virus being tested for, each tissueculture is assayed for ATP content by contacting a sample of it, in thepresence of oxygen, with firefly lantern extract and measuring theamount of light emitted. Preferably, the tissue cultures are assayed atperiodic intervals after one has been inoculated with the unknownmaterial. Comparisons of ATP content are made on the basis of ATP perunit tissue volume or ATP per unit protein weight or both.

The practice of this invention may be used to detect the presence of avirus in materials such as filtered air, drinking water, tissue samples,throat swabbings, excretions from lesions, etc. Samples of suchmaterials may be obtained in conventional fashion.

When a material is suspected to contain a virus but it is not known whattype of virus may be present, the practice of this invention may be usednot only to confirm the presence of a virus but also to indicate thetype of virus. This may be accomplished by inoculating several differenttypes of tissue cultures, each of which is a host for a different groupof viruses. Each of the types of tissue cultures is then inoculated withthe material suspected to contain a virus, incubated and assayed aspreviously described. It the results show that one of the types oftissue cultures has undergone a significant change in ATP content, thepresence of a virus will be confirmed and it will be established thatthe virus is one of the group for which that particular tissue cultureis a host.

The specific type of virus may be determined by providing equal aliquotsof the host tissue culture and adding a different anti-serum, each ofwhich is specific for a different virus, to each aliquot. Each aliquotis then inoculated with the material containing a virus. Following asuitable incubation period, the aliquots are assayed for ATP content aspreviously described. If the results show thatone tissue culture has notundergone a significant change in ATP content, the unknown virus will beidentified as the one for which the anti-serum contained in that tissueculture is specific.

In assaying the tissue cultures, results may be obtained by mixing theintact tissue cells with the firefly lantern extract. However, formaximal response and in order to make more accurate assays, it ispreferred to rupture the cells and extract the ATP therefrom. A varietyof methods for the extraction of ATP from the cells may be used. Theseinclude hot Water extraction, acetone extraction, ultrasonic disruption,dimethylsulfoxide extraction and perchloric acid extraction. Some of themethods which may be used for accomplishing rupture and extraction ofthe cells are described below:

(A) Acetone and hot water: One ml. of the tissue culture is added to 10ml. of deionized water and the suspension maintained at C. for one tofive minutes. The preparation is then cooled and assayed for ATP.

The acetone extraction consists of adding one ml. of a washed tissueculture suspension to ten ml. of acetone. After standing for one to fiveminutes, a one ml. aliquot of the mixture is taken to dryness in air andthe residue suspended in one ml. of deionized water. The preparation isthen assayed.

(B) Dimethyl sulfoxide (DMSO): DMSO is a highboiling point organicsolvent, miscible with water in all proportions and exhibiting a verylow order of toxicity. One ml. aliquots of the tissue cell suspensionare added to ten ml. of various concentrations of DMSO in water. Afterstanding for five minutes, the suspension is assayed for ATP response.

(C) Ultrasonic oscillation: Ultrasonic oscillation has been successfullyused by many investigators for the rupture of cells. Five ml. of tissuecell suspension are subjected to one to five minutes of ultrasonicoscillation at approximately 57-75 watts. After cooling the tube inflowing water, the treated suspension is assayed for ATP response.Trichloroacetic acid (0.5 ml. of percent solution) may be added to thecell suspension prior to sonification in order to stabilize the ATPagainst hydrolysis.

(D) Perchloric acid: Up to 0.2 ml. of perchloric acid may be added to 5ml. of tissue cell suspension. The preparation is then assayed.

It is preferred to contact the tissue culture to be assayed and thefirefly lantern extract in a liquid reaction medium. The liquid reactionmedium will generally contain enough dissolved oxygen to allow thebioluminescent reaction to take place.

The material to be assayed should be mixed with the firefly lanternextract in a manner which permits the mechanical measurement andrecordation of the light emitted.

The procedure for using the instruments which are used to detect andrecord the intensity of emitted light consists of injecting a liquidmedium containing the material to be assayed, such as an aqueous extractof the material, into a cuvette containing the firefly lantern extract.The extract is held at pH 7.4 with potassium arsenate buffer. The lightemitted as the result of the reaction between the ATP in the tissueculture to be tested and the firefly lantern extract strikes the surfaceof a photomultiplier tube giving rise to a current which can be measuredand recorded by either an oscilloscope photograph or a linear recorder.The unit of intensity used for comparing these reactions is themillivolt. Alternately, a pulse counting device with a digital oranalogue read-out may be used to record the reaction.

Because the response (i.e., light emission) is almost instantaneous whenthe tissue culture is contacted with the firefly lantern extract, theextract should be positioned in front of the light detection systemprior to the introduction of the material to be assayed.

There are two ways in which the bioluminescent response with ATP presentin a material can be expressed. One is by measurement of the maximumintensity of the emitted light, which after reaching this maximum value,decays exponentially. With all other factors constant, the maximumintensity is directly proportional to the concentration of ATP. Thealternative manner of expressing the response is by integration of thetotal amount of light emitted; i.e., area under the light intensitycurve. This is the slower of the two methods, because of the relativelylong time necessary for complete decay (up to minutes). Therefore,maximum intensity has been chosen as the measure of ATP response.

The instrumentation necessary for the quantitative measurement ofbioluminescence consists of a photomuliplier tube for the conversion oflight energy into an electrical signal, a device for determining themagnitude of the signal, and a light-tight chamber for presentation ofthe bioluminescent reaction to the photomultiplier tube.

In one system, part of the assembly consists of a composite sensing andreaction chamber which contains a photomultiplier tube, with appropriatecircuitry, and a rotary cylinder mounted in a block of aluminum in amanner which permits removal of the reaction chamber without exposingthe phototube to light. A section of the cylinder wall is cut out toaccommodate a standard'ten mm. or or five mm. rectangular cuvette.Immediately above the cuvette holder is a small injection port sealedwith a replaceable light-tight rubber plug. The entire unit is paintedblack to reduce light reflection. The photomultiplier converts the lightenergy into an electrical signal. An oscilloscope which records themagnitude of the signal from the photomultiplier, is provided with amaximum sensitivity of 200 u.v./cm. of beam deflection which will allowan increase in system sensitivity by decreasing the bandwidth ordirectly reducing the noise level. There is a multiple switchingarrangement at the scope input which makes it convenient to adjust thesystem zeros and balances. The differential input to the scope providesa means to balance the dark current output of the phototube. Theresponse to the firefly luminescent system displayed on the oscilloscopescreen is recorded with a camera which mounts directly onto the front ofthe oscilloscope. To observe and record the reaction, the cuvettecontaining the necessary reagents is positioned in the cuvette carrierwithout exposing the phototube. Rotation of the carrier positions thecuvette in front of the phototube. The tissue culture is then addedthrough the injection port and the magnitude of the response is recordedby the camera.

A typical procedure for assaying a material according to the practice ofthis invent on utilizing electronic apparatus to detect and record theintensity of the bioluminescent reaction is described below:

PROCEDURE A One ml. of a 0.5 percent buttered aqueous solution ofcommercially available lyophilized firefly lantern extract is placedinto a cuvette which is then positioned in the light detection chamberof the type previously described. The extract contains luciferase,luciferin and magnesium. Sufficient dissolved oxygen for thebioluminescent reaction is present in the solution. A portion of thetissue culture to be assayed is subjected to ultrasonic vibration forone or more minutes. One tenth ml. of the suspension is then drawn intoa hypodermic syringe and immediately injected through the light-proofseal into the cuvette. The reaction reaches maximum light intensity inless than 0.5 second and then decreases exponentially for severalminutes. The entire procedure can be executed and recorded in less thantwo minutes.

In order to make quantitative determinations of the amount of ATPpresent, the instrument used to measure the light response may becalibrated using known concentrations of ATP. A calibration may beplotted by injecting 1/10 ml. portions of known concentrations of ATPthrough the light-proof seal into the cuvette by means of a hypodermicsyringe. The light response in millivolts is plotted against the ATPconcentration. A straight linear function is obtained. For example, ifthe response from 10 grams of ATP is 20,000 millivolts, that from l0 is2000 millivolts, etc.

The overall sensitivity and perhaps reliability of the bioluminescentreaction of the material to be tested may be increased by the conversionof other nucleotide phosphates which are present in tissues, such asadenosine diphosphate (ADP) and adenosine monophosphate (AMP), to ATP.This may be accomplished as described below by adding to the tissueculture certain phosphorylating enzymes. One such enzyme isphosphocreatine kinase.

Phosphocreatine kinase (10-30 units/mg.) is prepared in a concentrationof 0.4 mg./ml. in 0.05 M potassium arsenate buffer (pH 7.4) containingMgSO at a concentration of 10- and creatine phosphate at a concentrationof 0.1 mg./ml. One tenth ml. of this solution is added to 1 ml. of asolution being assayed for ADP. The mixture is allowed to incubate at 30C. for 30 An aqueous suspension of monkey kidney tissue is divided into10 ml. aliquots. Five of the aliquots are inoculated by adding theretoequal aliquots of monkey kidney tissue infected with an adenovirus. Bothsets of cultures are incubated for the times indicated in the followingtable. Periodically, an aliquot is sonically ruptured and assayed forATP as described in Procedure A, above. The results are set forth in thetable.

TABLE A Ineu- Response.

bation light time, units,

Material tested days mv.

Virus infected, inoculated- 45 Control, non-inoculated 0 50 Virusinfected 2 125 Contr 2 30 Virus infected- 4 480 Control 4 10 Virusinfected 10 820 Control l0 0 EXAMPLE 2 Chicken embryo tissue cultureswere grown and inoculated with 1/ 10, 1/1000, and 1/10,000 dilutions ofinfluenza virus, A-2, strain lap 305. The infected and uninfectedcontrol cultures were incubated at 36 C. for 24 ohurs. After incubation,the fluid culture medium was removed and the tissue cell layer suspendedin 1 ml. of cold 0.02 M arsenate buffer at pH 7.4. The suspension wasadded to a centrifuge tube and 5 ml. of cold n-butanol added. Aftermixing for seconds the tubes were centrifuged at 500 g for 2 minutes.Aliquots 0.01 ml.) of the aqueous phase were removed and assayed for ATPusing procedure A as described above. The results are shown in Table B.

TABLE B Sample: Response (light units), mv. No virus 1,170 Virus,1/l0,000 dilution 1,150 Virus, 1/ 1000 dilution 1,120

Virus, 1/10 dilution 455 As can be seen, as the amount of virusincreased, the amount of bioluminescence decreased.

EXAMPLE 3 Chicken embryo tissue cultures were inoculated with SimlikiForest Virus at dilutions of 1/ 100 and l/ 10,000. The infected anduninfected control cultures were incubated at 36 C. for 6 hours and 24hours. After in cubation, the fluid culture medium was removed and thetissue cell layer suspended in 5.0 ml. of cold 0.02 M arsenate buffer atpH 7.4. The suspension Was centrifuged at 500 g for 2 minutes and thesupernatant discarded. The packed tissue cells were resuspended in coldbuffer and 1.0 ml. filtered through a bacterial filter. The cells werewashed with 1.0 ml. of cold buffer, treated with nbutanol and washedagain with 1.0 ml. of cold buffer. The extracted solution wascentrifuged at 500 g for 2 minutes and 0.01 ml. aliquot of the aqueouslayer removed for ATP assay. The results of the assay are shown in TableC.

The procedure as set forth in Example 3 is followed, except Hela tissueculture cells were used and were in fected with rhino virus. After 24hours incubation, the cells were analyzed for ATP, using above-describedprocedure A with the following results:

TABLE D Response, mv. HeLa cells, no virus 30,900 HeLa cells, +rhinovirus 25,600

EXAMPLE 5 Rabbit kidney cells were grown and inoculated with Herpessimplex virus, overlayed with an agar medium and incubated for 24 hours.After treatment similar to that described with respect to Example 4, theextracted ATP was measured with the following results:

TABLE E Response, mv. Rabbit kidney, no virus 71 Rabbit kidney, Herpesvirus 119 The comparison of the above examples illustrate that asignificant change (e.g. increase or decrease) in the amount ofbioluminescence given off by a sample indicates the presence of a virustherein. No significant change, of course, indicates the absence ofvirus.

EXAMPLE 6 The process of Example 1 is repeated except that the materialused to inoculate the tissues is a unknown suspected to contain anadenovirus. After a three day period of incubation, the bioluminescentassay indicates that the inoculated culture contains about 10 times asmuch ATP as the non-inculated culture. Thus, it is established that theunknown material contains a virus.

EXAMPLE 7 Ten ml. aqueous suspensions of each of the following tissuecultures are prepared: chicken embryo, hamster kidney, monkey kidney andHeLa cells. Each of the tissue cultures is inoculated with a 1 ml.portion of water which is suspected to contain a virus. Each tissueculture is incubated for a period of several days. Before incubation andperiodically during and following incubation, equal aliquots of eachtissue culture are sonically ruptured and assayed for ATP as describedin Procedure A, above. It is found that the ATP content of the chickenembryo tissue culture changed materially during incubation. The ATPcontent of all of the other tissue cultures did not changesignificantly. Thus, it is established that the water tested contains avirus that will reproduce in chicken embryo tissue culture but whichwill not reproduce in hamster kidney tissue cluture, monkey kidneytissue culture or HeLa culture.

EXAMPLE 8 An aqueous suspension of chicken embryo tissue culture isdivided into four 10 ml. aliquots. To each of three of the aliquotsthere is added a different specific antiserum for each of the followingviruses: St. Louis encephalitis, yellow fever and influenza. All fouraliquots are then inoculated by adding thereto 1 ml. portions of thewater tested in Example 7. Each tissue culture is then incubated forseveral days. Before incubation and periodically during and followingincubation, equal aliquots of each of the four suspensions of tissuecultures are sonically ruptured and assayed for ATP as described inProcedure A, above. It is found that the ATP contents of the controlculture (i.e., the one not containing an antiserum) and of the culturescontaining anti-Serums for St. Louis encephalitis and influenzaincreased several fold during incubation. However, the ATP content ofthe culture containing anti-serum for yellow fever does not increasesignificantly following incubation. This indicates that the water testedcontains yellow fever virus.

I claim:

1. A method for determining the presumptive presence of a virus in anunknown material which comprises inoculating a tissue culture which isan appropriate host for the suspected virus with a sample of thematerial suspected to contain a virus, incubating said inoculated tissueculture and measuring and comparing the ATP content of said tissueculture both before and after said incubation period, the results ofsaid comparison indicating the presence or absence of a virus in saidmaterial.

2. The method of claim 1 wherein said ATP content is measured by mixingequal aliquots of said tissue culture, one before and one after saidincubation period, with firefly lantern extract, said extract containingluciferin, luciferase and magnesium, and measuring the amount of lightemitted.

3. A method for determining the presumptive presence of a virus in anunknown material which comprises providing duplicate tissue cultureswhich are hosts for the suspected virus, inoculating one of said tissuecultures with a sample of the unknown material suspected to contain avirus, after an incubation period mixing each of said tissue cultures inthe presence of oxygen with firefly lantern extract, said extractcontaining luciferin, luciferase and magnesium, measuring the amount oflight emitted and comparing the amount of light emitted by theinoculated tissue culture with the amount of light emitted by thenon-inoculated tissue culture, the results of said comparison indicatingthe presence or absence of a virus in said material.

4. The method of claim 3 wherein said virus is adenovirus and whereinsaid host tissue is monkey kidney tissue.

5. The method of claim 3 wherein said virus is yellow fever virus andsaid tissue culture is chicken embryo.

6. A method for determining the presumptive presence of a virus in ahost tissue which comprises providing duplicate tissue cultures whichare hosts for the suspected virus, inoculating one of said tissuecultures with a sample of the host tissue suspected to contain a virus,incubating said tissue cultures, at periodic intervals treating equalportions of each of said tissue culture to rupture the cells of saidtissue and extract the adenosine triphosphate therefrom and thereaftermixing said treated cultures in the presence of oxygen with fireflylantern extract, said extract containing luciferin, luciferase andmagnesium, measuring the amount of light emitted and comparing theamount of light emitted by the inoculated tissue culture with the amountof light emitted by the noninoculated tissue culture, the results ofsaid comparison indicating the presence or absence of a virus in saidhost tissue.

7. A method for determining the presumptive presence of a virus in anunknown material which comprises providing a plurality of differenttissue cultures, each tissue culture being a host for a different groupof viruses, inoculating each tissue culture with a sample of the unknownmaterial suspected to contain a virus, both before and after anincubation period mixing equal aliquots of each of said tissue culturesin the presence of oxygen with firefly lantern extract, said extractcontaining luciferin, luciferase and magnesium, measuring the amount oflight emitted and comparing the amount of light emitted by eachinoculated tissue culture before and after incubation, the results ofsaid comparison indicating the presence or absence of virus in saidmaterial and indicating which one, if any, of said tissue cultures is ahost for any virus which is present.

8. The method of claim 7 wherein said plurality of tissue culturescomprise chicken embryo, hamster kidney, monkey and HeLa cells and saidvirus is one which causes an increase in ATP after said incubation onlyin said chicken embryo.

9. A method for identifying the type of virus present in a materialwhich comprises providing a plurality of equal aliquots of a tissueculture which is a host for said virus, adding a different anti-serum toeach of said aliquots, each of said anti-serums being specific for adifferent virus, inoculating each tissue culture with the virus,incubating each of said tissue cultures, both before and after saidincubation mixing equal amounts of each of said tissue cultures in thepresence of oxygen with firefly lantern extract, said extract containingluciferin, luciferase and magnesium measuring the amount of lightemitted and comparing the amount of light emitted by each inoculatedtissue culture before and after incubation, the results of saidcomparison indicating the type of virus present in said material.

10. The method of claim 9 wherein said tissue culture is chicken embryoand said virus is yellow fever virus.

References Cited UNITED STATES PATENTS 3,117,061 1/1964 Grafe 103.5

ALVIN E. TANENHOLTZ, Primary Examiner US. Cl. X.R. 1951.l, 66

